Epigenetics offers many targets that could revolutionize cancer therapy. Systemic delivery of epigenetic inhibitors is unfeasible due to deleterious effects on regenerating tissue. Delivering inhibitors with cancer- specific bacteria would conquer this critical problem by specifically focusing treatment to tumors and metastases. To date, no one has produced bacteria that modulate epigenetic targets. Bacterial delivery systems would function by transporting peptides and shRNA into cancer cells. Inhibiting the key targets EZH2, NIPP1 and PP1 would disrupt essential cancer cell processes and eliminate cancer stems cells. This will stop tumor spreading and prevent metastasis formation. The proposed research has four Specific Aims that combine the newest developments in the fields of epigenetics and bacterial cancer targeting. Aim 1 will create a bacterial macromolecule delivery system; Aim 2 will create a bacterial shRNA delivery system that targets NIPP1 and EZH2; Aim 3 will create a bacterial peptide delivery system that disrupts NIPP1:PP1 complexes; and Aim 4 will create failsafe circuitry to clear bacteria after treatment. The effect of gene knockdown and complex disruption will be tested by target-protein measurement, immunoprecipitation, and phosphatase phosphorylase assay. Cell viability and the cancer stem cell populations will be quantified in a tumor-on-a-chip device, subcutaneous tumors and spontaneous metastases in mice. The groundwork for this study has been established by 1) creating a non-toxic bacterial macromolecule delivery system, 2) demonstrating its key components (intracellularly triggered lysis and cytoplasmic localization) and its ability to delive proteins and DNA; 3) validating NIPP1 and EZH2 knockdowns in cancer cells and tumors; and 4) showing that bacterial release of NIPP1:PP1 dissociative peptides induces cancer cell death. The proposed research will build upon a collaboration between Neil Forbes (Chemical Engineering, UMass Amherst, USA), and Aleyde Van Eynde and Mathieu Bollen (Cellular and Molecular Medicine, KU Leuven, Belgium), experts in bacterial cancer treatment and epigenetic regulators. This research will establish a new therapeutic platform for controlled gene and protein delivery into cancer cells. Transient delivery of genes and proteins with bacteria will enable direct targeting of proteins and functions specifically in cancer cells. This study will create an epigenetic therapy with the potential to eradicate metastases and prevent their formation, two urgent clinical problems. Proposed animal experiments will be the first to demonstrate the benefit of a protein-phosphatase-directed therapy that disrupts protein-protein interactions in holoenzymes. The ultimate goal of this project is development of a treatment modality for primary cancers and metastatic disease.